ABSTRACT Osteoarthritis (OA) is the most prevalent joint disease. Although many drug targets have been identified that were successful in preclinical studies, clinical trials on disease modifying OA drugs thus far have failed and pain management remains unsatisfactory. Approaches such as testing disease-related differences in expression of selected genes or proteins and analyzing their function in preclinical models has yielded a large number of pathways and molecules that are abnormal in OA. The limitations of these approaches are (i) that they provide only a selective view of molecular changes in OA and (ii) there has been no successful effort in integrating these findings into networks and prioritizing targets by their relevance as drivers of the OA process. This project leverages (i) our access to and expertise in working with human knee tissues from donors across the entire adult age spectrum and at all stages of OA development; (ii) existing and growing human knee tissue libraries; (iii) technical advances in genome wide analyses of transcriptomic changes, which provide an unbiased and comprehensive view of the genetic landscape of cartilage homeostasis and OA; (iv) our expertise in developing pipelines for integrative network analysis of multi-Omics data sets. Our hypothesis is that dysregulation transcription factors (TFs) is a major determinant of the abnormal gene expression pattern that drives OA pathogenesis. Our approach is to generate data from gene expression (mRNAseq) and enhancer activation analysis (GRO-seq) at the tissue level and more precisely at the single cell level to identify novel signatures, pathways and key regulators of cartilage homeostasis and OA. Aim 1. The transcriptomic landscape of normal and OA human articular cartilage single cell levels. We will perform single-cell RNAseq to identify chondrocyte subpopulations in normal and OA human articular cartilage. Aim 2. Enhancer profiling to identify drivers of pathogenic gene expression patterns in OA. Active enhancers are characterized by the presence of enhancer RNAs (eRNAs). We will use GRO-seq to assay eRNA transcription in normal and OA chondrocytes. These results will reveal pathways and networks that are disrupted in OA and identify principal regulators of OA pathogenesis. Aim 3. Validation: Confirm differences in TF expression and activation in joint tissues and analyze function in joint tissue cells. We will assess differences in TF protein expression and activity in cartilage and other joint tissues and determine the role of candidate TFs in mediating expression of OA-associated gene patterns. Impact: To our knowledge, this is the first project to examine genome-wide mRNA expression profiles in healthy and OA-affected knee cartilage at tissue and single cell levels and linking this transcriptomic data with analysis of TF expression and activity. The study has potential discover novel pathways and principal molecular switches as therapeutic targets. Ultimately this may lead to interventions to delay or treat OA.